Journal: bioRxiv

Article Title: Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

doi: 10.1101/2025.01.08.631865

Figure Lengend Snippet: (A) Quantification of IST amplitude in bRG cells following treatment with DMSO, nocodazole (1 μ M) or blebbistatin (10 μ M) in cortical organoids (N=3 organoid batches, 329 bRG cells, weeks 9-12). (B) Live imaging of mitotic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (week 8-11). shRNA plasmids co-express GFP. (C) Live imaging of mitotic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (pcw 16-20). (D) Live imaging of in vitro interphasic human bRG cells expressing control or LIS1 shRNA constructs. (E) Quantification of IST amplitude in in vitro interphasic human bRG cells expressing control or LIS1 shRNA constructs (N=3 experiments, 520 bRG cells). (F) Quantification of IST amplitude in in vitro interphasic human bRG cells expressing control or KASH constructs, in the presence of DMSO or blebbistatin (10 μ M) (N=3 experiments, 1198 bRG cells). Yellow arrowheads indicate bRG cell soma, and green and red arrowheads indicate daughter cells. Data are presented as mean values +/− SD. Scale bar = 20 µm. All live imaging montages are in hours:minutes. **p<0,01; ***p<0,001, ns: non-significant by two-tailed unpaired t-tests.

Article Snippet: The following plasmids were used in this study: MSCV-IRES-GFP (Tannishtha Reya, Addgene 20672); VSVG (a gift from P. Benaroch), Human EZR shRNA (TF308420, Origene), Human STK10 shRNA (TF320540, Origene), Human SLK shRNA (TG320620, Origene), Human DYNC1H1 shRNA (TL313335, Origene), Human RDX shRNA (TL309884, Origene), Human MSN shRNA (TL311375, Origene), Human ECT2 shRNA (TL304854, Origene), Human VIM shRNA (TL308419, Origene), Human PAFAH1B1 (LIS1) shRNA (TL310628, Origene), Dominant Negative KASH.

Techniques: Imaging, Expressing, Control, shRNA, Construct, In Vitro, Two Tailed Test

Journal: bioRxiv

Article Title: Two independent translocation modes drive neural stem cell dissemination into the human fetal cortex

doi: 10.1101/2025.01.08.631865

Figure Lengend Snippet: (A) Live imaging of interphasic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (week 8-11). shRNA plasmids co-express GFP. (B) Live imaging of interphasic human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (pcw 16-18). (C) Quantification of IST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (N=3 organoid batches, 899 bRG cells, week 8-11). Two independent shRNA plasmids were used for each knockdown. (D) Quantification of MST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human cortical organoids (N=3 organoid batches, 899 bRG cells, week 8-11). (E) Quantification of IST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (N=3 fetal samples, 385 bRG cells, pcw 16-20). (F) Quantification of MST amplitude in human bRG cells expressing control, DYNC1H1 or LIS1 shRNA constructs in human fetal tissue (N=3 fetal samples, 385 bRG cells, pcw 16-20). (G) Live imaging of interphasic human bRG cells expressing GFP in control cortical organoids and two different patient-derived LIS1-mutated organoids (week 8-11). (H) Quantification of IST amplitude in control cortical organoids and two different patient-derived LIS1-mutated organoids (N=3 organoid batches, 397 bRG cells, week 8-11). (I) Quantification of MST amplitude in control cortical organoids and two different patient-derived LIS1-mutated organoids (N=3 organoid batches, 397 bRG cells, week 8-11). (J) Immunostaining for SOX2 and Nesprin-2 in cortical organoids expressing GFP or the KASH dominant negative together with GFP (week 9). Red arrows indicate nuclear envelope of construct-expressing cells. (K) Live imaging of interphasic human bRG cells expressing control or KASH constructs in human cortical organoids (week 8). KASH plasmid co-expresses GFP. (L) Live imaging of interphasic human bRG cells expressing control or KASH constructs in human fetal tissue (pcw 16). KASH plasmid co-expresses GFP. (M) Quantification of IST amplitude in human bRG cells expressing control or KASH constructs in human cortical organoids (N=3 organoid batches, weeks 8-11, 201 bRG cells). (N) Quantification of MST amplitude in human bRG cells expressing control or KASH constructs in human cortical organoids (N=3 organoid batches, weeks 8-11, 201 bRG cells). (O) Quantification of IST amplitude in human bRG cells expressing control or KASH constructs in human fetal tissue (N=2 fetal samples, pcw 16-18, 40 bRG cells). (P) Quantification of MST amplitude in human bRG cells expressing control or KASH constructs in human fetal tissue (N=2 fetal samples, pcw 16-18, 40 bRG cells). Yellow arrowheads indicate bRG cell soma, and green and red arrowheads indicate daughter cells. Data are presented as mean values +/− SD. Scale bar = 20 µm. All live imaging montages are in hours:minutes. **p<0,01; ****p<0,0001, ns: non-significant by two-tailed unpaired t-tests.

Article Snippet: The following plasmids were used in this study: MSCV-IRES-GFP (Tannishtha Reya, Addgene 20672); VSVG (a gift from P. Benaroch), Human EZR shRNA (TF308420, Origene), Human STK10 shRNA (TF320540, Origene), Human SLK shRNA (TG320620, Origene), Human DYNC1H1 shRNA (TL313335, Origene), Human RDX shRNA (TL309884, Origene), Human MSN shRNA (TL311375, Origene), Human ECT2 shRNA (TL304854, Origene), Human VIM shRNA (TL308419, Origene), Human PAFAH1B1 (LIS1) shRNA (TL310628, Origene), Dominant Negative KASH.

Techniques: Imaging, Expressing, Control, shRNA, Construct, Knockdown, Derivative Assay, Immunostaining, Dominant Negative Mutation, Plasmid Preparation, Two Tailed Test

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: Antibody reagents

Article Snippet: Hs00322286_m1 , DYNC1H1.

Techniques: Western Blot, Immunohistochemistry-IF

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: TaqMan assays

Article Snippet: Hs00322286_m1 , DYNC1H1.

Techniques:

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: Sequences of primers used in qPCR

Article Snippet: Hs00322286_m1 , DYNC1H1.

Techniques: Sequencing

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: SiRNA reagents

Article Snippet: Hs00322286_m1 , DYNC1H1.

Techniques:

Journal: Scientific Reports

Article Title: Identification of a novel gene signature for the prediction of recurrence in HCC patients by machine learning of genome-wide databases

doi: 10.1038/s41598-020-61298-3

Figure Lengend Snippet: Vascular invasion-related genes.

Article Snippet: OBSCN in TCGA database, PLXNA1, MUC12 and BSN in AMC database, and BIRC6, DNAH5, PKHD1, TSC2, KIAA1109 and DYNC1H1 in Inserm database were detected.

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Evidence for dynein and astral microtubule–mediated cortical release and transport of Gα i /LGN/NuMA complex in mitotic cells

doi: 10.1091/mbc.E12-06-0458

Figure Lengend Snippet: Gα i -regulated interaction between LGN and DYNC1H1. (A) Endogenous LGN and cytoplasmic dynein form a complex. MDCK II cells were partially synchronized by treating with nocodazole (200 nM) for 12 h. After being released from the treatment for 40 min, cells were harvested and subjected to immunoprecipitation using anti-LGN antibodies or rabbit IgG. The immunoprecipitates were separated by SDS–PAGE and blotted using specific antibodies. (B) Gα i enhances the association between LGN and DYNC1H1. Cos 7 cells were transfected as indicated. Cell lysates (1/30 of total) and immunoprecipitates were separated in a 6% SDS–PAGE gel and blotted using specific antibodies. Note that in 6% gel, myc-Gα i 1 comigrated with and was masked by the light chain of anti-HA antibody in the HA-LGN immunoprecipitates. (C) Gα i , LGN, and DYNC1H1 form a complex in vivo, independent of NuMA. Cos 7 cells were transfected as indicated. Cell lysates (1/30 of total) and immunoprecipitates were separated in a 7% SDS–PAGE gel and blotted. (D) The GoLoco-insensitive Gα i 1 cannot associate with LGN and DYNC1H1. Cos 7 cells were transfected as indicated. Cell lysates were subjected to analysis as in C.

Article Snippet: Full-length human DYNC1H1 cDNA was transferred from KIAA cDNA clone (0325; Kazusa DNA Research Institute, Kisarazu, Japan) by PCR and restriction enzyme digestion and cloned in pK-VENUS ( Du et al. , 2001 ).

Techniques: Immunoprecipitation, SDS Page, Transfection, In Vivo

Journal: Molecular Biology of the Cell

Article Title: Evidence for dynein and astral microtubule–mediated cortical release and transport of Gα i /LGN/NuMA complex in mitotic cells

doi: 10.1091/mbc.E12-06-0458

Figure Lengend Snippet: LGN is required for the cortical localization of DYNC1H1 during mitosis. (A) LGN depletion results in reduced cortical localization of DYNC1H1. MDCK cells transduced by control lentivirus (control) or lentivirus expressing shRNAs targeting different regions of LGN (LGN-KD1-7 and LGN-KD2-6) were stained with anti-DYNC1H1 antibody and DNA dye. Bar, 10 μm. (B) Quantitation of the fluorescence intensity of cortical DYNC1H1 from images acquired in A. n = 50 for each set; * p < 0.01. (C) Slight overexpression of LGN leads to enhanced cortical localization of DYNC1H1. Stable Tet-Off MDCK cells expressing Venus-LGN were cultured in the presence (+Dox) or absence (–Dox) of doxycycline. At 24 h later, cells were stained as in A. Bar, 10 μm. (D) Quantitation of the fluorescence intensity of cortical DYNC1H1 from images acquired in C. n = 50 for each set; * p < 0.01.

Article Snippet: Full-length human DYNC1H1 cDNA was transferred from KIAA cDNA clone (0325; Kazusa DNA Research Institute, Kisarazu, Japan) by PCR and restriction enzyme digestion and cloned in pK-VENUS ( Du et al. , 2001 ).

Techniques: Expressing, Staining, Quantitation Assay, Fluorescence, Over Expression, Cell Culture

Journal: Molecular Biology of the Cell

Article Title: Evidence for dynein and astral microtubule–mediated cortical release and transport of Gα i /LGN/NuMA complex in mitotic cells

doi: 10.1091/mbc.E12-06-0458

Figure Lengend Snippet: Knocking down DYNC1H1 or disrupting astral MTs leads to enhanced cortical localization of LGN. (A) MDCK cells were transfected with plasmids expressing control shRNA (control) or shRNAs targeting DYNC1H1 (shRNA1 and shRNA2). At 48 h later, cells were fixed and stained with anti-LGN, anti–α-tubulin antibodies, and DNA dye. Bar, 10 μm. (B) MDCK cells were cultured in media containing 50 nM nocodazole for 40 min. Cells were then fixed and stained as in A. Bar, 10 μm. (C) Quantitation of the fluorescence intensity of cortical LGN from images acquired in A and B. n = 50 for each set; * p < 0.01.

Article Snippet: Full-length human DYNC1H1 cDNA was transferred from KIAA cDNA clone (0325; Kazusa DNA Research Institute, Kisarazu, Japan) by PCR and restriction enzyme digestion and cloned in pK-VENUS ( Du et al. , 2001 ).

Techniques: Transfection, Expressing, shRNA, Staining, Cell Culture, Quantitation Assay, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Evidence for dynein and astral microtubule–mediated cortical release and transport of Gα i /LGN/NuMA complex in mitotic cells

doi: 10.1091/mbc.E12-06-0458

Figure Lengend Snippet: Dynamic turnover of cortical LGN relies on astral MTs and DYNC1H1. (A) FRAP analysis of cortical LGN. Representative images from live-cell time-lapse series were shown. The photobleaching areas were marked by squares. Stable MDCK cells expressing Venus-LGN were either untreated (top), treated with 50 nM nocodazole for 1 h (middle), or transfected with DYNC1H1 shRNA for 48 h (bottom) before being subjected to FRAP analysis. (B–E) Quantitative analysis of FRAP experiments. (B, D) Plots of normalized fluorescence intensity of cortical Venus-LGN in cells treated with DMSO (B, blue diamonds), 50 nM nocodazole (B, pink squares), or transfected with control shRNA (D, blue diamonds) or DYNC1H1 shRNA (D, pink squares) vs. time (in seconds) after photobleaching. Data are expressed as mean ±SEM ( n = 8 for each set). (C, E) The mobile fractions of cortical Venus-LGN in control, nocodazole-treated, or DYNC1H1-knockdown cells were calculated from the fluorescence recovery curves shown in B and D. Data are expressed as mean ±SEM. * p < 0.01. (F) Association of Venus-LGN with astral MTs. MDCK cells expressing Venus-LGN were preextracted with microtubule stabilization buffer containing 0.2% Triton X-100 and then fixed with 4% PFA. Fixed cells were stained with anti-LGN (green), anti–α-tubulin (red) antibodies, and DNA dye (blue). Bar, 10 μm.

Article Snippet: Full-length human DYNC1H1 cDNA was transferred from KIAA cDNA clone (0325; Kazusa DNA Research Institute, Kisarazu, Japan) by PCR and restriction enzyme digestion and cloned in pK-VENUS ( Du et al. , 2001 ).

Techniques: Expressing, Transfection, shRNA, Fluorescence, Staining

Journal: Molecular Biology of the Cell

Article Title: Evidence for dynein and astral microtubule–mediated cortical release and transport of Gα i /LGN/NuMA complex in mitotic cells

doi: 10.1091/mbc.E12-06-0458

Figure Lengend Snippet: Disruption of actin filaments leads to astral microtubule– and dynein-dependent cortical dissociation and spindle pole accumulation of LGN. (A) MDCK II cells were either untreated (Control) or treated as labeled. LatA: 1 μM of LatA for 45 min; Nocodazole + LatA: 50 nM of nocodazole plus 1 μM of LatA for 45 min; DYNC1H1 shRNA-1, -2 + LatA: transfected with DYNC1H1 shRNA-1 or -2 for 48 h and then treated with 1 μM LatA for 45 min. MG132, 5 μM, was added 1 h before treatments and maintained during treatments. Cells were fixed after treatments and stained with anti-LGN (green), anti–α-tubulin (red) antibodies, and DNA dye (blue). (B) Quantitation of LGN signals at spindle poles as described in Materials and Methods . n = 50 for each set; * p < 0.01.

Article Snippet: Full-length human DYNC1H1 cDNA was transferred from KIAA cDNA clone (0325; Kazusa DNA Research Institute, Kisarazu, Japan) by PCR and restriction enzyme digestion and cloned in pK-VENUS ( Du et al. , 2001 ).

Techniques: Labeling, shRNA, Transfection, Staining, Quantitation Assay

Journal: Molecular Biology of the Cell

Article Title: Evidence for dynein and astral microtubule–mediated cortical release and transport of Gα i /LGN/NuMA complex in mitotic cells

doi: 10.1091/mbc.E12-06-0458

Figure Lengend Snippet: Disruption of actin filaments leads to astral microtubule–dependent cortical dissociation and spindle pole accumulation of DYNC1H1 and NuMA. (A) Venus-LGN–expressing cells were treated as in except for the results at the bottom, for which cells were first treated with 1 μM LatA for 45 min and then treated with 1 μM LatA plus 50 nM nocodazole for another 45 min. Cells were stained with anti-DYNC1H1 antibody (red) and DNA dye (blue). (B) Quantitation of cortical DYNC1H1 fluorescence intensity as described in Materials and Methods . n = 50 for each set; * p < 0.01. (C) Venus-LGN–expressing cells were treated as in A. Cells were stained with anti-NuMA antibody (red) and DNA dye (blue). Second from top, arrow points to the original crescent-shaped NuMA, and arrowhead points to NuMA accumulated at the spindle pole. (D) Quantitation of cortical NuMA fluorescence intensity. n = 50 for each set; * p < 0.01. Bars, 10 μm.

Article Snippet: Full-length human DYNC1H1 cDNA was transferred from KIAA cDNA clone (0325; Kazusa DNA Research Institute, Kisarazu, Japan) by PCR and restriction enzyme digestion and cloned in pK-VENUS ( Du et al. , 2001 ).

Techniques: Expressing, Staining, Quantitation Assay, Fluorescence